Biological low-molecular-weight derivatives

ABSTRACT

The crosslinking agents and condensing agents that have been employed in biological adhesives and in treating medical devices such as cardiac valves are non-natural compounds synthesized artificially. Thus, they are not metabolized in vivo and exhibit toxicity to living bodies. These compounds are thus used only in a restricted amount and for limited purposes in the clinical sites. The present invention provides a biological low-molecular-weight derivative obtained by modifying carboxyl groups of a biological low-molecular-weight compound with N-hydroxysuccinimide, N-hydroxysulfosuccinimide, or a derivative thereof and a crosslinked high-molecular-weight product obtained by crosslinking various high-molecular-weight compounds with this derivative.

TECHNICAL FIELD

The present invention relates to biological low-molecular-weightderivatives obtained by modifying carboxyl groups of biologicallow-molecular-weight compounds having two or more carboxyl groups withN-hydroxysuccinimide, N-hydroxysulfosuccinimide, or derivatives thereof,and to crosslinked high-molecular-weight products synthesized using thebiological low-molecular-weight derivatives.

BACKGROUND ART

In biological adhesives and in treating medical devices that are derivedfrom biological compounds, such as porcine cardiac valves, crosslinkingagents containing artificially and chemically synthesized aldehydes suchas glutaraldehyde or condensing agents such as1-ethyl-3-(3-dimethylaminopropyl)carbodiimide have been used (forexample, refer to patent documents 1 to 6, and non-patent document 1).

-   Patent document 1: Japanese Unexamined Patent Application    Publication No. 7-163650-   Patent document 2: Japanese Unexamined Patent Application    Publication No. 9-249751-   Patent document 3: Japanese Unexamined Patent Application    Publication No. 10-71199-   Patent document 4: PCT Japanese Translation Patent Publication No.    2000-502380-   Patent document 5: Japanese Unexamined Patent Application    Publication No. 8-53548-   Patent document 6: PCT Japanese Translation Patent Publication No.    8-502082-   Non-patent document 1: Biomaterials, vol. 17, p. 765 (1996)

DISCLOSURE OF INVENTION

Most crosslinking agents and condensing agents that have been used totreat medical devices and the like are non-natural, artificiallysynthesized products. Thus, they are not metabolized in vivo and exhibittoxicity to living bodies. They are thus used in limited amounts and forlimited purposes in clinical sites. In order to overcome such problems,development of biological crosslinking agents is desired.

The present invention provides a biological low-molecular-weightderivative obtained by modifying carboxyl groups of a biologicallow-molecular-weight compound having two or more carboxyl groups withN-hydroxysuccinimide, N-hydroxysulfosuccinimide, or a derivativethereof, and a crosslinked high-molecular weight product synthesizedusing the biological low-molecular-weight derivative.

In detail, the present invention provides a biologicallow-molecular-weight derivative obtained by modifying at least onecarboxyl group of a biological low-molecular-weight compound having twoor more carboxyl groups with N-hydroxysuccinimide,N-hydroxysulfosuccinimide, or a derivative thereof. This biologicallow-molecular-weight derivative is harmless to human bodies and achievesfast reaction since two or more reactive groups (—COOH) are contained.

The present invention also provides a crosslinked high-molecular weightproduct prepared by crosslinking a high-molecular-weight compound with abiological low-molecular-weight derivative obtained by modifying atleast one carboxyl group of a biological low-molecular-weight compoundhaving two or more carboxyl groups with N-hydroxysuccinimide,N-hydroxysulfosuccinimide, or a derivative thereof.

When this crosslinked high-molecular weight product is applied to livingbodies, the compound is metabolized in vivo, and is absorbed anddisappears after a predetermined time. Thus, no extraneous matterremains in the body.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chart showing a nuclear magnetic resonance spectrum of acitric acid derivative.

BEST MODE FOR CARRYING OUT THE INVENTION

A biological low-molecular-weight compound having two or more carboxylgroups used in the present invention is a tri- or dicarboxylic acidlow-molecular-weight compound in the citric acid cycle. Examples of thetri- or dicarboxylic acid low-molecular-weight compound include malicacid, oxalacetic acid, citric acid, cis-aconitic acid, 2-ketoglutaricacid, and derivatives thereof.

The biological low-molecular-weight derivative of the present inventionis obtained by reacting carboxyl groups of the biologicallow-molecular-weight compound having two or more carboxyl groups withN-hydroxysuccinimide, N-hydroxysulfosuccinimide, or a derivative thereofhaving low cytotoxicity in the presence of carbodiimide to introduceactive esters.

Such a compound is obtained by reacting 0.001 to 10 percent by weight ofthe biological low-molecular-weight compound with 0.001 to 10 percent byweight of N-hydroxysuccinimide, N-hydroxysulfosuccinimide, or aderivative thereof in the presence of 0.001 to 20 percent by weight ofcarbodiimide (EDC) at a suitable reaction temperature in the range of 0Cto 100° C. and a reaction time in the range of 1 to 48 hours.

Examples of the carbodiimide include1-ehtyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC),1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate,and dicyclohexylcarbodiimide. Examples of the reaction solvent includeN,N-dimethylformamide (DMF) and dimethylsulfoxide (DMSO).

FIG. 1 is a chart showing a nuclear magnetic resonance spectrum of acitric acid derivative yielded by the reaction between citric acid andN-hydroxysuccinimide in the presence of EDC. The peak a shows themethylene proton of citric acid and the peak b shows the methyleneproton of the succinimidyl group. The remaining two peaks areattributable to the solvent (DMSO).

Examples of the protein used in preparing the crosslinked compoundinclude collagen (any of several ten types), atelocollagen (any one ofseveral ten types), alkali-soluble collagen (any of several ten types),gelatin, keratin, serum albumin, egg albumin, hemoglobin, casein, andamino-containing polymers such as globulin and fibrinogen. Theseproteins may be derived from any organism.

Examples of glycosaminoglycans used to prepare the crosslinked productinclude chondroitin sulfate, dermatan sulfate, hyaluronic acid, heparansulfate, heparin, keratan sulfate, and their derivatives. Theglycosaminoglycans may have any molecular weight and may be derived fromany organism.

Examples of other high-molecular-weight compounds include chitosan (thedegree of deacetylation and molecular weight are not limited), polyaminoacids (the type of amino acid and molecular weight are not limited), andpolyalcohols (the type and molecular weight are not limited).

The crosslinking reaction between the biological low-molecular-weightderivative and the high-molecular-weight compound is conducted byreacting 0.1 to 50 percent by weight of the high-molecular-weightcompound with 0.01 to 50 percent by weight of the biologicallow-molecular-weight derivative at preferably 30° C. to 50° C. These twocompounds are preferably mixed as solutions having predeterminedconcentrations to facilitate synthesis of a homogeneous crosslinkedproduct. Examples of the solvents used to prepare such solutions includenontoxic solvents such as distilled water, buffer solutions, e.g.,physiological saline, sodium hydrogen carbonate, boric acid, andphosphoric acid, and organic solvents (DMF, DMSO, and ethanol).

EXAMPLES Example 1-1

To a 5 wt % DMF solution of citric acid, 3.2 equivalents ofN-hydroxysuccinimide and 3.1 equivalents of EDC were added at roomtemperature, and the resulting mixture was stirred for 24 hours.Subsequently, only the DMF, i.e., the organic solvent in the reactionsolution, was removed by reduced-pressure distillation. The residue waspurified with an acetone/n-hexane developing solvent by chromatographyover a silica gel column to synthesize a derivative having threecarboxyl groups of citric acid modified with N-hydroxysuccinimide.

Example 1-2

The synthesis of collagen gel obtained by the reaction scheme belowusing the citric acid derivative synthesized in Example 1-1 will now bedescribed:

The synthesized citric acid derivative (24 μL ) was dissolved in 976 μLof a dimethylsulfoxide solution. A 100 μL portion of this solution wasweighed and added to 400 μL of a 1.25 wt % phosphoric acid buffersolution of type II collagen. The resulting mixture was stirred and leftto stand still for 24 hours at room temperature to obtain a collagen gelhaving a crosslinking agent concentration of 0.4 to 40 mM. The gel wasweighed, dried in a freeze-dry machine, and weighed again to determinethe water content of the gel. The water content of the gel is shown inTable 1. TABLE 1 Concentration of citric acid derivative (mM) Watercontent (%) 0.4 98 1 98 2 96 4 97 8 97 10 97 20 98 30 98 40 98

Example 2-1

To a 5 wt % DMF solution of 2-ketoglutaric acid, 2.2 equivalents ofN-hydroxysuccinimide and 2.1 equivalents of EDC were added at roomtemperature. The resulting mixture was stirred for 24 hours.Subsequently, only the DMF, i.e., the organic solvent in the reactionsolution, was removed by reduced-pressure distillation. The residue waspurified with an acetone/n-hexane developing solvent by chromatographyover a silica gel column to obtain a derivative having two carboxylgroups of 2-ketoglutaric acid modified with N-hydroxysuccinimide.

Example 2-2

The synthesis of collagen gel obtained by the reaction scheme belowusing the 2-ketoglutaric acid derivative synthesized in Example 2-1 willnow be described:

The synthesized 2-ketoglutaric acid derivative (14 μL) was dissolved in986 μL of a dimethylsulfoxide solution. A 100 μL portion of thissolution was weighed and added to 400 μL of a 1.25 wt % phosphoric acidbuffer solution of type II collagen. The resulting mixture was stirredand left to stand still for 24 hours at room temperature to obtain acollagen gel having a crosslinking agent concentration of 0.6 to 10 mM.The gel was weighed, dried in a freeze-dry machine, and weighed again todetermine the water content of the gel. The water content of the gel isshown in Table 2. TABLE 2 Concentration of 2-ketogluraic acid derivative(Mm) Water content (%) 0.6 98 0.8 98 1 98 4 97 8 98 10 97

Example 3-1

To a 5 wt % DMF solution of cis-aconitic acid, 3.2 equivalents ofN-hydroxysuccinimide and 3.1 equivalents of EDC were added at roomtemperature. The resulting mixture was stirred for 24 hours.Subsequently, only the DMF, i.e., the organic solvent in the reactionsolution, was removed by reduced-pressure distillation. The residue waspurified with an acetone/n-hexane developing solvent by chromatographyover a silica gel column to obtain a derivative having three carboxylgroups of cis-aconitic acid modified with N-hydroxysuccinimide.

Example 3-2

The synthesis of collagen gel obtained by the reaction scheme belowusing the cis-aconitic acid derivative synthesized in Example 3-1 willnow be described:

The synthesized cis-aconitic acid derivative (46 μL) was dissolved in954 μL of a dimethylsulfoxide solution. A 100 μL portion of thissolution was weighed and added to 400 μL of a 1.25 wt % phosphoric acidbuffer solution of type II collagen. The resulting mixture was stirredand left to stand still for 24 hours at room temperature to obtain acollagen gel having a crosslinking agent concentration of 1 to 30 mM.The gel was weighed, dried in a freeze-dry machine, and weighed again todetermine the water content of the gel. The water content of the gel isshown in Table 3. TABLE 3 Concentration of cis-aconitic acid derivative(mM) Water content (%) 1 97 2 97 4 97 8 97 10  97 30  97

Example 4-1

To a 5 wt % DMF solution of malic acid, 2.2 equivalents ofN-hydroxysuccinimide and 2.1 equivalents of EDC were added at roomtemperature. The resulting mixture was stirred for 24 hours.Subsequently, only the DMF, i.e., the organic solvent in the reactionsolution, was removed by reduced-pressure distillation. The residue waspurified with an acetone/n-hexane developing solvent by chromatographyover a silica gel column to obtain a derivative having two carboxylgroups of malic acid modified with N-hydroxysuccinimide.

The synthesis of collagen gel obtained by the reaction scheme belowusing the malic acid derivative synthesized in Example 4-1 will now bedescribed:

The synthesized malic acid derivative (32 μL) was dissolved in 968 μL ofa dimethylsulfoxide solution. A 100 μL portion of this solution wasweighed and added to 400 μL of a 1.25 wt % phosphoric acid buffersolution of type II collagen. The resulting mixture was stirred and leftto stand still for 24 hours at room temperature to obtain a collagen gelhaving a crosslinking agent concentration of 3 to 50 mM. The gel wasweighed, dried in a freeze-dry machine, and weighed again to determinethe water content of the gel. The water content of the gel is shown inTable 4. TABLE 4 Concentration of malic acid derivative (mM) Watercontent (%) 3 97 4 98 5 97 6 97 8 97 10  97 20  97 40  97 50  97

Example 5-1

To a 5 wt % DMF solution of oxalacetic acid, 2.2 equivalents ofN-hydroxysuccinimide and 2.1 equivalents of EDC were added at roomtemperature. The resulting mixture was stirred for 24 hours.Subsequently, only the DMF, i.e., the organic solvent in the reactionsolution, was removed by reduced-pressure distillation. The residue waspurified with an acetone/n-hexane developing solvent by chromatographyover a silica gel column to obtain a derivative having two carboxylgroups of oxalacetic acid modified with N-hydroxysuccinimide.

Example 5-2

The synthesis of collagen gel obtained by the reaction scheme belowusing the oxalacetic acid derivative synthesized in Example 5-1 will nowbe described:

The synthesized oxalacetic acid derivative (16 μL) was dissolved in 984μL of a dimethylsulfoxide solution. A 100 μL portion of this solutionwas weighed and added to 400 μL of a 1.25 wt % phosphoric acid buffersolution of type II collagen. The resulting mixture was stirred and leftto stand still for 24 hours at room temperature to obtain a collagen gelhaving a crosslinking agent concentration of 2 to 40 mM. The gel wasweighed, dried in a freeze-dry machine, and weighed again to determinedthe water content of the gel. The water content of the gel is shown inTable 5. TABLE 5 Concentration of oxalacetic acid derivative (mM) Watercontent (%) 2 98 4 97 6 98 8 97 10  98 20  96 40  98

INDUSTRIAL APPLICABILITY

The gelate biological high-molecular-weight product described above isapplied to one of biological adhesives, hemostatic agents, materials forembolizing blood vessels, and sealing materials for aneurysum to performcrosslinking reaction directly at affected sites. The compound may becrosslinked in advance and then be applied to adhesion preventingagents, scaffolds for tissue regeneration, and drug carriers.

1. A biological low-molecular-weight derivative obtained by modifying atleast one carboxyl group of a biological low-molecular-weight compound,which is selected from the group consisting of malic acid, oxalaceticacid, citric acid, cis-aconitic acid, and derivatives thereof, has twoor more carboxyl groups, and is in the citric acid cycle, withN-hydroxysuccinimide, N-hydroxysulfosuccinimide, or a derivativethereof, wherein the biological low-molecular-weight derivativeundergoes hydrolyzation in vivo after application in vivo and reactswith a biological high-molecular-weight compound.
 2. (canceled) 3.(canceled)
 4. A crosslinked high-molecular-weight product obtained bycrosslinking a high-molecular-weight compound with the biologicallow-molecular-weight derivative according to claim 1, the crosslinkedhigh-molecular-weight product comprising a gel that is metabolized invivo after application in vivo.
 5. The crosslinked high-molecular-weightproduct according to claim 4, wherein the high-molecular-weight compoundis at least one of proteins, glycosaminoglycans, chitosans, polyaminoacids, and polyalcohols.
 6. The crosslinked high-molecular-weightproduct according to claim 4, wherein the high-molecular-weight compoundis a glycosaminoglycan comprising chondroitin sulfate, dermatan sulfate,hyaluronic acid, heparan sulfate, heparin, keratan sulfate, or aderivative thereof.
 7. The crosslinked high-molecular-weight productaccording to claim 4, wherein the high-molecular-weight compound is aprotein comprising collagen, atelocollagen, alkali-soluble collagen,gelatin, keratin, serum albumin, egg albumin, hemoglobin, casein,globulin, fibrinogen, or a derivative thereof.
 8. The crosslinkedhigh-molecular-weight product according to claim 4, wherein the productis applied to one of biological adhesives, hemostatic agents, materialsfor embolizing blood vessels, and sealing materials for aneurysum toperform crosslinking reaction directly at affected sites.
 9. Thecrosslinked high-molecular-weight product according to claim 4, whereinthe product is applied to one of adhesion preventing agents, scaffoldsfor tissue regeneration, and drug carriers after performance ofcrosslinking reaction.
 10. A method for producing the biologicallow-molecular-weight derivative according to claim 1, comprisingreacting 0.001 to 10 percent by weight of a biologicallow-molecular-weight compound, which is selected from the groupconsisting of malic acid, oxalacetic acid, citric acid, cis-aconiticacid, 2-ketoglutaric acid, and derivatives thereof, has two or morecarboxyl groups, and is in the citric acid cycle, with 0.001 to 10percent by weight of N-hydroxysuccinimide, N-hydroxysulfosuccinimide, ora derivative thereof in the presence of 0.001 to 20 percent by weight ofcarbodiimide at a reaction temperature of 0° C. to 100° C. for areaction time of 1 to 48 hours to modify at least one carboxyl group ofthe biological low-molecular-weight compound with N-hydroxysuccinimide,N-hydroxysulfosuccinimide, or a derivative thereof.
 11. A method forproducing a crosslinked high-molecular-weight product comprisingcrosslinking a high-molecular-weight compound with a biologicallow-molecular-weight derivative obtained by the method according toclaim 10 so as to yield a crosslinked high-molecular-weight compoundcomprising a gel that is metabolized in vivo after application in vivo.